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They were derived from the human embryonic kidney line, 293 (see ATCC CRL-1573).

The CMV promoter was excised (EcoRI/XhoI, blunt-ended), and replaced with the MoMLV LTR (Asp 718/HindIII, blunt-ended) from plasmid pVH2.

The amphotropic envelope-encoding plasmid, pCMVEa, was transfected into ProGag cells and clones yielding high transduction efficiencies were isolated. One of these, clone number 52 (ProPak-A.52), was deposited as CRL-12479.

They were derived from the human embryonic kidney line, 293 (see ATCC CRL-1573).

To construct the ProPak-X and the ProPak-A-52 cell lines, the ATG in the splice donor/splice acceptor of pCMV plasmid was mutated to ACG.

The CMV promoter was excised (EcoRI/XhoI, blunt-ended), and replaced with the MoMLV LTR (Asp 718/HindIII, blunt-ended) from plasmid pVH2.

The beta-galactosidase gene was replaced by the gag-pol ORF (NotI fragment) to generate pMoMLVgp. pMoMLVgp was co-transfected with pHA58 into 293 cells by calcium phosphate co-precipitation and hygromycin B-resistant cells were selected.

Clones were screened for the level of Gag secretion and one clone secreting high levels of Gag was selected (designated ProGag); this clone yielded high viral titers in transient transfection.

The amphotropic envelope-encoding plasmid, pCMVEa, was transfected into ProGag cells and clones yielding high transduction efficiencies were isolated. One of these, clone number 52 (ProPak-A.52), was deposited as CRL-12479.

ProPak-based producer cells were demonstrated to be free of replication-competent retrovirus (RCR) by stringent testing.

The highest transduction of human hematopoietic progenitor cells was achieved with vector supernatant generated from a coculture of the ProPak-X and ProPak-A cell lines.

The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.

Subculturing

Protocol: Remove medium. Do not rinse. Cells detach easily. Add 2.0 to 3.0 ml of 0.25% trypsin, 0.53 mM EDTA solution. Allow the flask to sit at room temperature (or at 37C) until the cells detach. Centrifuge the cell suspension at 1000 rpm for 10 minutes, resuspend the pellet in fresh medium, aspirate and dispense into new flasks.

Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:10 is recommended

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